In recent years, cis- and trans-RNA elements have become well recognized as important regulators of gene expression. Cells use diverse non-coding RNA-based elements to regulate complex genetic networks such as those involved in developmental timing and circadian clocks (Banerjee et al., Bioessays 24, 119-29 (2002); and Kramer et al., Nature 421, 948-52 (2003)). Antisense RNAs are small trans-acting RNAs (taRNAs) that bind to complementary segments of a target messenger RNA (mRNA) and regulate gene expression through mechanisms such as targeting decay, blocking translation, and altering splicing patterns (Good, Cell Mol Life Sci 60, 823-4 (2003); Good, Cell Mol Life Sci 60, 854-61 (2003); and Vacek et al., Cell Mol Life Sci 60, 825-33 (2003)). MicroRNAs (miRNAs), small taRNAs that affect either translation or RNA decay by interacting with complementary sequences in mRNA and the genome, are likely widespread in metazoan gene regulation (Bartel, Cell 116, 281-97 (2004)). Small interfering RNAs (siRNAs) and double-stranded RNAs (dsRNAs) are able to precisely target mRNAs and inhibit their expression through the RNA interference (RNAi) pathway in metazoans, and are thought to be part of the cell's host defense system (Scherer, Curr Pharm Biotechnol 5, 355-60 (2004)). Ribozymes are RNA molecules exhibiting catalytic function and have been shown to be used by viruses to regulate gene expression (Lilley, Trends Biochem Sci 28, 495-501 (2003)). Riboswitches, cis-acting metabolite binding structures in mRNAs, control gene expression by modulating translation initiation, disruption of transcriptional termination, or cleavage of mRNA by ribozyme mechanisms (Mandal et al., Nat Struct Mol Biol 11, 29-35 (2004); Winkler, Nature 419, 952-6 (2002); and Winkler, Nature 428, 281-6 (2004)). Recent studies have demonstrated the prevalence of these RNA-based regulators across diverse groups of organisms from prokaryotes to humans (Barrick et al., Proc Natl Acad Sci USA 101, 6421-6 (2004); Yelin et al., Nat Biotechnol 21, 379-86 (2003); and Lavorgna et al., Trends Biochem Sci 29, 88-94 (2004)).
Researchers have taken advantage of the relative ease with which RNA libraries can be generated and searched to create synthetic RNA-based molecules with novel functional properties. Aptamers are nucleic acid binding species that interact with high affinity and specificity to selected ligands. These molecules are generated through iterative cycles of selection and amplification known as in vitro selection or SELEX (Systematic Evolution of Ligands by EXponential enrichment) (Ellington et al., Nature 346, 818-22 (1990); and Tuerk et al., Science 249, 505-10 (1990)). Aptamers have been selected to bind diverse targets such as dyes, proteins, peptides, aromatic small molecules, antibiotics, and other biomolecules (Hermann et al., Science 287, 820-5 (2000)). High-throughput methods and laboratory automation have been developed to generate aptamers in a rapid and parallel manner (Cox et al., Nucleic Acids Res 30, e108 (2002)). Researchers have demonstrated that aptamers can impart allosteric control properties onto other functional RNA molecules. Such allosteric control strategies have been employed to construct and select in vitro signaling aptamers, in vitro sensors, and in vitro allosterically controlled ribozymes (Jhaveri et al., Nat Biotechnol 18, 1293-7 (2000); Roth et al., Methods Mol Biol 252, 145-64 (2004); and Stojanovic et al., J Am Chem Soc 126, 9266-70 (2004)).
In addition to the widespread occurrence of RNA-based regulator elements in natural systems, researchers have recently described engineered riboregulator systems. Cis-acting RNA elements were described that regulate relative expression levels in Escherichia coli from a two gene transcript by controlling RNA processing and decay (Smolke et al, Appl Environ Microbiol 66, 5399-405 (2000)). In another example, a combined cis/trans riboregulator system was described in E. coli in which cis-acting RNA elements mask the ribosome binding site of a transcript, thereby inhibiting translation, and trans-activating RNAs bind to the cis-acting elements to allow translation (Isaacs et al., Nat Biotechnol 22, 841-7 (2004)). Cis-acting elements were recently described that control gene expression in mammalian cells and mice by acting through RNA cleavage and whose activity can be regulated by a small molecule drug and antisense oligonucleotides (Yen et al., Nature 431, 471-6 (2004)). Finally, an allosteric aptamer construct was recently described that upon binding the dye tetramethylrosamine, interacts with protein-based transcriptional activators to induce transcription (Buskirk et al., Chem Biol 11, 1157-63 (2004)).
Riboregulators present powerful tools for flexible genetic regulation. However, there is a need to couple the ability of RNA-based regulators that can directly target transcripts with allosteric control typically associated with protein-based regulators.